A naturally occuring protein isolated from the saliva of the medicinal leech Hirudo medicinalis is described which strongly binds to collagen thus acting as an inhibitor of natural platelet adhesion to collagen. The protein has a molecular weight of about 12 000, an acidic isoelectric point and contains six cysteins. The protein was sequenced and the gene was cloned from a H. medicinalis cDNA-library. Procedures for producing such polypeptide by recombinant techniques are disclosed. The recombinant and the natural occuring proteins are potent inhibitors of collagen-dependent platelet adhesion and therefore useful for the therapeutic treatment of various conditions related to hard disease and diseases of the circulation system. Furthermore the protein is useful for coating natural or artificial collagen surfaces in order to render them nonadhesive for cells and prevent the activation of cells.
In haemostasis or thrombosis platelets adhere to the cell-extracellular matrix of an injured vessel and cover the surface of the damaged area. Preventing this important initial step in the pathogenesis of thrombosis and arterial occlusion should be of therapeutic benefit in the effort of to prevent thrombotic diseases. Collagen is considered to be the most thrombogenic surface component and has been shown to be a strong stimulant for platelet adhesion, aggregation and the release of their granules leading to the recruitment of (Ruggeri, Z. M. et al.; Seminars in Hematology, 1994, 31, 229-39) additional platelets to this area to form aggregates or a thrombus. The initial contact of the platelets to the vessel surface is mediated by collagen bound van Willebrand Factor (vWF) and a specific vWF receptor on platelets, the glycoprotein Ib-V-IX complex. In addition ADP, epinephrine and circulating clotting factors drive the further activation process of platelets while simultaneously an increase in thrombin activity contributes to the formation of the cross-linked fibrin clot. Platelet-platelet aggregation supports this process and is mainly driven by fibrinogen as a mediator that bridges cells through the glycoprotein IIb/IIIa receptor.
This normal physiological response is quite critical in the course of the pathological process where platelets adhere to collagenes exposend in sclerotic lesions (Van der Rest M. et al.; FASEB Journal, 1991, 5, 2814-23) and start to bild-up occlusions. Depending on the location and extent of the occlution severe complications such as myocardial infarction, stroke, inflammation or pulmonary embolism may be the severe outcome of this process.
As a direct acting antithrombotic agent heparin which blocks the thrombin activity, thus preventing the formation of fibrin rich thrombi, is the currently most well known drug used in anti-thrombotic interventions. Heparin is widely used in indications such as: unstable angina and acute myocardial infarction. However despite the wide use several severe short comings such as intravenous application, requirement for anti-thrombin-III as a cofactor, reduced affinity for clot-bound thrombin, it""s inactivation by several plasma proteins, the occasional induction of thrombocytopenia and it""s biological heterogeneity remain unresolved. As a consequence the results of using heparin in the clinical setting have not been overvelming sofar.
Recent development of low molecular weight heparin has contributed a version for subcutaneous application, however the therapeutic benefit over the standard heparin has been modest. Unfortunately the same applies to the other directly acting antithrombins such as Hirudin, Hirulog and Warfarin. It turned out, that one of the major problems seems to be related to the increased production of thrombin under antithrombotic treatment (Rao, A. K et al., Circulation, 1996, 94, 389-2395).
Other recent strategies have therefore been focussed to the process of prothrombin activation which is driven by Factor Xa. The major challenge is the design of appropriate inhibitors directed to this factor. In summary on would therefore argue that the full therapeutic potential of this type of intervention has not yet been realized.
Another pannel of therapeutics is represented by the thrombolytic regimens and has been focussed on the development of staphylokinase, streptokinase, urokinase type Plasminogen Activator, tissue type Plasminogen Activator and anisoylated-plasminogen-streptokinase activator complex. The differences in time necessary to inducing reperfusion is remarkable different for each of these thrombolytic agents, however the contribution in terms of reducing the overall mortality is equal for all the products. In addition reocclusion or prolonged bleeding are frequent complications. This might be due to relatively low specificity for fibrin and the short plasma half-life of these compounds. Currently various application regimens and combinations of different fibrinolytic principles are tested in order to overcome some of the current short comings in thrombolytic therapie. The improvement which are expected are however rather small.
Recently a new group of patients occured with probelms such as acute thrombotic occlusion and late restenosis due to procedures such as angioplasty, atherectomy, arterial grafting or vessel wall stenting. The possible therapeutic interventions comprise anti-platelet, antithrombotic and thrombolytic strategies. Various other agents such as ticlopdine acting as ADP antagonists or Calcium ionophore A-23187 and especially Asprin have a direct influence on the platelet function and have been suggested or used to prevent or minimize platelet aggregation. The new anti-platelet adhesion substance according to this invention could as well help to overcome these clinical complications when applied during surgery.
Another complication related to this topic arises if artificial surfaces come in contact with blood, then their is increased tendency to induce thrombotic events by activation of platelets and/or induction of coagulation. These effects may cause failure of vascular grafts, cardiac valves, stents, catheters or any other blood contacting device or material. The ability of the protein disclosed here to create non-thrombogenic surfaces may therefore be further exploited by immobilization of this protein to the materials and devices described above Such a treatment should render such materials or devices biocompatible and thromboresistant.
Due to the limitations associated with the available antithrombotic agents there is an actual need for new alternative strategies and therapeutics.
A potential for future improvements in the treatement of caridvacular disorders may be contributed by approaches as disclosed in this invention which directely interfere with the collagen and/or vWF factor induced platelet adhesion.
Several novel inhibitors which prevent platelet adhesion are monoclonal to antibodies directed to vWF. It has as well been suggested that glycoprotein IIb/IIIa inhibitors may be beneficial in inhibiting platelet adhesion.
Some of these inhibitors like the monoclonal Ab c7E3 have already been tested clinically while others like the KGD- and RGDF-inhibitors are still under study. However, the specificity of most of these new inhibitors is not very well studied, thus the spectrum of side effects that will be induced by using this inhibitors is still open and deserves carefull examination.
A rich source for the screening of new compounds that interfere with collagen induced platelet adhesion is given in nature through blood-sucking animals. Several inhibitors have been isolated from nature as described in the literature: A 65 kD protein called Calin isolated from Hirudo medicinalis (U.S. Pat. No. 5,587,360 WO 92/07005) (Munro, R. et al., Blood Coagulation and Fibrinolysis, 1991, 2, 179-184) and a 16 kD (LAPP) protein isolated from the salivary glands of the leech Haementera officinalis (U.S. Pat. No. 5,324,715). Both proteins have been described as aggregation inhibitors as tested in static assays of collagen dependent platelet aggregation.
Despite a proven in vitro activity LAPP failed to act in several well-established in vivo models (Schaffer L. W. et al., Arterioscler. Thromb., 1993, 13, 1593-1601) and Connolly T. M. et al.; Thromb. Haemostas., 1993, 69, 589). The soft tick, Omithodoros moubata, also contains an antiplatelet protein (Moubatin) which is active in preventing collagen-stimulated platelet aggregation (Waxman, L. et al.; J. Biol. Chem., 1993, 268, 5445-49). Another inhibitor of platelet aggregation from a blood-sucking bug was disclosed in WO 9309137 by Noeske-Jungblut C. et al. Smith et al. have isolated a 50 kDa protein from snake venom and a 19 kDa protein was isolated from a the saliva of Triatoma pallidipennis, a blood-sucking bug. The protein was found to contain a factor that specifically inhibits collagen-induced platelet aggregation. The 19 kDa protein named pallidipin inhibits collagen-mediated aggregation of platelets in plasma. No inhibition of aggregation stimulated by other effectors (ADP, thrombin, thromboxane A2 mimetic U46619, phorbol ester) was detected. Pallidipin had no effect on platelet adhesion to collagen but inhibited ATP release from platelets. It interacted reversibly with platelets and may share with collagen a common target on them. The precise mechanism of action and therapeutic benefit of this protein is under investigation. Gan et al. described Echistatin as an inhibitor binding to the fibrinogen receptor GP IIa/IIIb (J. Biol. Chem, 1988, 263, 19827-32).
Despite these exciting developments, the need continues to exist for supplying further anticoagulants and antithrombin which have increased efficacy in the inhibition of clot formation, vWF-induced platelet activation or endothelial cell activation and which may be used pharmaceutically and produced in commercially feasible quantities.
Since none of the known proteins described sofar has developed into a compound with ideal therapeutic profile the inventors of the present invention decided to go ahead with a new screening strategy in order to detect more relevant proteins.
In this invention an inhibitor isolated from H. medicinalis is described which directly acts one collagen-platelet interaction thus inhibiting platelet activation and early platelet-platelet interaction.
So far, there has not been a positive example in the literature which indicates that by using a screening approach that would exclude aggregation inhibitors as well as lytic proteins from a source of naturally occurring compounds one could identify new anti-adhesive mechanisms or compounds. However this strategy was used in this invention. Since at least six different platelet surface glycoproteins are known to be involved in collagen adhesion and in addition several platelet derived compounds such as von Willebrand factor, fibronectin and thrombospondin have been shown to be involved as indirect mediators of collagen-platelet adhesion there has been little optimism in the beginning to identity a new adhesion inhibitor.
Nevertheless, this approach was used to screen Hirudo medicinalis saliva knowing that not all the documented or unkown vWF related inhibitors as well as compounds directely acting on platelet receptors could be excluded. Thus the result of the screening was a surprise: A new protein named Saratin with anti-adhesive activity for plateles which can be isolated from tissues and secretions of well investigated leech of the species Hirudo medicinalis. 
The present invention comprises the active polypeptide Saratin isolated from the leech Hirudo medicinalis. The protein was isolated from saliva by a combination of pressurized dialysis and at least one chromatographic step like anionic exchange chromatography and at least one reversed phase high performance chromatography (RP-HPLC) step. The pressure dialysis step turned out to be absolutely critical during the recovery of Saratin from saliva, since the strong concentration of saliva helped to overcome the otherwise tremendous loss of bioactive Saratin. The isolated Saratin binds strongly to several collagens and blocks the adhesion of platelets to collagen coated such surfaces in a dose dependent fashion.
In order to optimize the screening cascade currently available techniques have been developed to distinguish platelet adhesion and platelet aggregation: the ability of platelets to retard or stop flow through fibers, the contribution of platelets to in vitro clot formation, glass bead adhesion laboratories, or whole blood flows through the filter and platelet adhesion of anticoagulated platelet-rich plasma to filters composed of glass fibers or collagen under a regulated pressure gradient.
The protein (named Saratin) is characterized by the amino acid sequences depicted in sequence (SEQ. ID. NO. 2) and is constituted from 103 amino acids which make up a theorectical relative molecular weight of approximately 12068 dalton ∓1 kDa The protein exhibits a unique primary structure with no significant similarity to other previously described sequences. The protein is rich in aspartic and glutamic acid which contribute to the low isoelectric point of of pH 3.7xc2x10.5 of the molecule as measured by IEF-PAGE.
SDS-PAGE analysis demonstrated a strong shift in mobility upon reduction of the protein proir to electrophoresis, indicating posttranslational modifications. Sequencing of the polypeptide had revealed six cysteine molecules which could make-up post-translational modifications of the protein. Electrospray mass spectrometry of Saratin revealed an actual molecular weight of 12061 indicating that up to three disulphide bonds are involved in the formation of the secondary structure of the native form of the protein.
The adhesion inhibitor according to the present invention is new, because it differs from known aggregation inhibitors isolated from leeches especially from Calin or LAPP in the molecular weight, isoelectric point and amino acid sequence and biological activities.
The present invention provides as well isolated DNA comprising a polynucleotide encoding the leech derived platelet adhesion inhibitor having the amino acid sequence as shown for the protein. The nucleotide sequence representing the cDNA clone is shown in SEQ. ID. NO. 1. Position 1-63 of the nucleotide sequence represents a putative 21 amino acid leader sequence and position 64-372 contains an open reading frame coding for a polypeptide of 103 amino acid residues and an amino acid sequence as shown for the mature protein in SEQ. ID. NO. 2.
The present invention also relates to recombinant vectors which include the synthetic gene coding for the leech-derived platelet adhesion inhibitor of the present invention, and a host cell containing the recombinant vectors. Methods for recovering and isolating the expressed proteins have been based on tag-technologies or have been adapted from the purification scheme developed for the naturally occuring Saratin. Depending on the individual protocols used for extracellular or intracellular expression in yeast cells, insect cells, baby hamster kidney cells and E. coli cells transformed with the appropriate vectors the steps for recovering the recombinant protein from the supernatant or sediments have to be adapted by techniques known to the expert. Excellent expression was found in E. coli as a host, where periplasmatic expression was contributed by insertion of a pelB leader sequence. Products recovery from Escherichia coli (E. coli) was achieved (arround 5 mg/l) after osmolysis and centrifugation. Saccharomyces cerevisiae (S. cerevisiae) ( greater than 10 mg/l culture broth) with the alterative yeast adopted vector was used in a paralelled experiment. The secreted material was isolated by centrigfugration. Purification was achieved by cross-flow filtration and ion exchange chromatography. In other expression approches using either COS cells or CHO cells product expression was arround 750 ng/ml. The purified recombinant material proved to be pure and homogeneous by electrophoretic and chromatographic analysis and identical to saliva derived Saratin as demonstrated by amino acid sequencing and molecular mass determination.
The invention also comprises methods for purifying the active protein from crude saliva of the leech and measuring its activity against platelets by static and dynamic assay methods as well as the use of this method to isolate rekombinant protein.
Techniques for the production of Saratin, include the Examples 6, 7, 8 and 13 however the expression methods to be used are not restricted to these examples. For instance transgenic mice, or other organisms, including other mammals, may as well be used to express Saratin.
Proteins of the present invention include variants which conserve the activity of the disclosed sequences, including fragments or subunits, naturally occuring variants, allelic variants, randomly generated artificial mutants and intentional sequence variations such as adding which conserve activity. Fragments or subunits refers to to any portion of the sequence which contain fewer amino acids than the complete protein e.g. partial sequences excluding portions of the N- and/or C-termini of the complete protein.
The invention further covers hybrid proteins, such as fusion proteins or proteins resulting from the expression of multiple genes within the expression vector, and may include a polypeptide having the specific activity of a disclosed protein linked by peptide bonds to a second polypeptide. Notably other variants of the proteins of the present invention are included, especially any variants that differ from the isolated protein only by conservative amino acid substitution. Such conservative amino acid substitutions are defined in Taylor et al., J. Mol. Biol., 1986, 188, 233.
Also included are methods for using the proteins to prevent or delay of platelet activation by inhibition of collagen-platelet interactions. The protein is useful in the prevention; prophylaxis, therapy and treatment of thrombotic diseases. Unlike all these previously described proteins, which act at various surface proteins on the platelet, the protein from this invention has a unique mechanism of action. It binds tightly to the collagen surface and one mechanism of action is given by the coverage of specific collagen sides no longer available for interactions and binding of platelets. This type of new mechanism has the great advantage, that the platelets stay functionally intact during the application of the protein, so that very low or even no bleeding tendency is expected from this treatment.
Another important area of use is the treatment of various surfaces with the protein to render them non-adhesive for platelets and thereby create blood-compatible devices.
As indicated above, the polypeptides according to the present invention are suitable as pharmaceutically effective compounds in parmaceutical compositions and combinations.
The pharmaceutical formulations according to the invention optionally may comprise additional active incredients like Aspirin, anti-coagulants such as hirudin or heparin or thrombolytic agents such as plasminogen activator or streptokinase.
The novel polypeptide according to the invention may form pharmaceutically acceptable salts with any non-toxic, organic or inorganic acid. Inorganic acids are, for example, hydrochloric, hydrobromic, sulphuric or phosphoric acid and acid metal salts such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate. Examples for organic acids are the mono, di and tri carboxylic acids such as acetic, glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, hydroxymaleic, benzoic, hydroxybenzoic, phenylacetic, cinnamic, salicylic and sulfonic acids such as methane sulfonic acid. Salts of the carboxy terminal amino acid moiety include the non-toxic carboxylic acid salts formed with any suitable inorganic or organic bases. These salts include, for example, alkali metals such as sodium and potassium, alkaline earth metals such as calcium and magnesium, light metals of Group IIIA including aluminium, and organic primary, secondary and tertiary amines such as trialkylamines, including triethylamine, procaine, dibenzylamine, 1-ethenamine, N,Nxe2x80x2-dibenzylethylene-diamine, dihydroabietylamine and N-alkylpiperidine.
As used herein, the term xe2x80x9cpharmaceutically acceptable carrierxe2x80x9d means an inert, non toxic solid or liquid filler, diluent or encapsulating material, not reacting adversely with the active compound or with the patient. Suitable, preferrably liquid carriers are well known in the art such as steril water, saline, aqueous dextrose, sugar solutions, ethanol, glycols and oils, including those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil and mineral oil.
The formulations according to the invention may be administered as unit doses containing conventional non-toxic pharmaceutically acceptable carriers, diluents, adjuvants and vehicles which are typical for parenteral administration.
The term xe2x80x9cparenteralxe2x80x9d includes herein subcutaneous, intravenous, intra-articular and intratracheal injection and infusion techniques. Also other administrations such as oral administration and topical application are suitable. Parenteral compositions and combinations are most preferably adminstered intravenously either in a bolus form or as a constant fusion according to known procedures.
Tablets and capsules for oral administration contain conventional excipients such as binding agents, fillers, diluents, tableting agents, lubricants, disintegrants, and wetting agents. The tablets may be coated according to methods well known in the art.
Oral liquid preparations may be in the form of aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for reconstitution with water or another suitable vehicle before use. Such liquid preparations may contain conventional additives like suspending agents, emulsifying agents, non-aqueous vehicles and preservatives.
Topical applications may be in the form of aqueous or oily suspensions, solutions, emulsions, jellies or preferably emulsion ointments.
Unit doses according to the invention may contain daily required amounts of the protein according to the invention, or sub-multiples thereof to make up the desired dose. The optimum therapeutically acceptable dosage and dose rate for a given patient (mammals, including humans) depends on a variety of factors, such as the activity of the specific active material employed, the age, body weight, general health, sex, diet, time and route of administration, rate of clearance, the object of the treatment, i. e., therapy or prophylaxis and the nature of the thrombotic disease to be treated, antiplatelet or anticoagulant activity.
Therefore, in compositions and combinations useful as antithrombotic in a treated patient (in vivo) a pharmaceutical effective daily dose of the peptides of this invention is between about 0.01 and 100 mg/kg body weight, preferably between 0.1 and 10 mg/kg body weight According to the application form one single dose may contain between 0.5 and 10 mg of the thrombin inhibitor To achieve an anticoagulant effect in extracorporeal blood a pharmaceutically effective amount of the inventive peptides is between 0.2 and 150 mg/l, preferably between 1 mg and 20 mg/l extracorporeal blood.
It is also object of this invention to provide an implantable or extracorporal medical device for use in contact with body fluids in order to render the device surface substantially thromboresistant, coated with an immobilized polypeptide as defined above and in the claims. The polypeptide according to the invention is immobilzed on a medical device so as to render the surface biocompatible and thromboresistant. Such devices sometimes have surfaces properties which typically induce platelet aggregation, which is a disadvantage in their intended uses in inplantable and extracorporeal devices in contact with blood or other body fluids. Examples for such devices which are commonly made from plastics materials and synthetic fibres are protheses, artificial organs, ocular lenses, sutures, artificial vascular segments, catheters, dialysers, tubes and vessels carrying blood.
Posterior capsule opacification (PCO) is a common complication after cateract extraxtion, despite the modem surgical techinques and lenses which are used for this procedure. PCO is caused by the proliferation and migration of lens epitheiial cells across the posterior capsule thus reducing the visual acuity. Physical treatments as well as chemically modified lenses have been proposed to reduce formation of PCO. Heparin lens coating or topical heparin eyedrops have been used to reduce PCO, indicating that thrombogenic mechanisms are involved in the formation of PCO.
Saratin has been shown to have significant advantages over heparin in preventing and blocking thrombogenicity. It is therefore another feature of this invention to provide a coating comprising Saratin which reduces thrombogenicity of the lens material used for refractive anterior or posterior chamber ocular implants which may be surgically implanted into the eye. This new type of coating avoids problems contributed by stimulated cell growth. In combination with other medicaments which are for instance confering cell death, Saratin coating-helps to completely overcome posterior capsule opacification.